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Project description:Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. Fifty-one candidates were selected in athree-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec). By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.
| S-EPMC4644809 | biostudies-literature
Project description:Rapid evolution of RNA viruses with mRNA-sense genomes is a major concern to health and economic welfare because of the devastating diseases these viruses inflict on humans, animals, and plants. To test whether host genes can affect the evolution of RNA viruses, we used a Saccharomyces cerevisiae single-gene deletion library, which includes approximately 80% of yeast genes, in RNA recombination studies based on a small viral replicon RNA derived from tomato bushy stunt virus. The genome-wide screen led to the identification of five host genes whose absence resulted in the rapid generation of new viral RNA recombinants. Thus, these genes normally suppress viral RNA recombination, but in their absence, hosts become viral recombination "hotbeds." Four of the five suppressor genes are likely involved in RNA degradation, suggesting that RNA degradation could play a role in viral RNA recombination. In contrast, deletion of four other host genes inhibited virus recombination, indicating that these genes normally accelerate the RNA recombination process. A comparison of deletion strains with the lowest and the highest recombination rate revealed that host genes could affect recombinant accumulation by up to 80-fold. Overall, our results demonstrate that a set of host genes have a major effect on RNA virus recombination and evolution.
| S-EPMC1180806 | biostudies-other
Project description:Pseudomonas fluorescens and related plant root ("rhizosphere")-associated species contribute to plant health by modulating defenses and facilitating nutrient uptake. To identify bacterial fitness determinants in the rhizosphere of the model plant Arabidopsis thaliana, we performed a high-throughput transposon sequencing (Tn-Seq) screen using the biocontrol and growth-promoting strain Pseudomonas sp. WCS365. The screen, which was performed in parallel on wild-type and immunocompromised Arabidopsis plants, identified 231 genes that increased fitness in the rhizosphere of wild-type plants. A subset of these genes decreased fitness in the rhizosphere of immunocompromised plants. We hypothesized that these genes might be involved in avoiding plant defenses and verified 7 Pseudomonas sp. WCS365 candidate genes by generating clean deletions. We found that two of these deletion mutants, ?morA (encoding a putative diguanylate cyclase/phosphodiesterase) and ?spuC (encoding a putrescine aminotransferase), formed enhanced biofilms and inhibited plant growth. We found that mutants ?spuC and ?morA induced pattern-triggered immunity (PTI) as measured by induction of an Arabidopsis PTI reporter and FLS2/BAK1-dependent inhibition of plant growth. We show that MorA acts as a phosphodiesterase to inhibit biofilm formation, suggesting a possible role in biofilm dispersal. We found that both putrescine and its precursor arginine promote biofilm formation that is enhanced in the ?spuC mutant, which cannot break down putrescine, suggesting that putrescine might serve as a signaling molecule in the rhizosphere. Collectively, this work identified novel bacterial factors required to evade plant defenses in the rhizosphere.IMPORTANCE While rhizosphere bacteria hold the potential to improve plant health and fitness, little is known about the bacterial genes required to evade host immunity. Using a model system consisting of Arabidopsis and a beneficial Pseudomonas sp. isolate, we identified bacterial genes required for both rhizosphere fitness and for evading host immune responses. This work advances our understanding of how evasion of host defenses contributes to survival in the rhizosphere.
| S-EPMC6222131 | biostudies-literature
Project description:Recent studies suggest progranulin (GRN) is a neurotrophic factor. Loss-of-function mutations in the progranulin gene (GRN) cause frontotemporal lobar degeneration (FTLD), a progressive neurodegenerative disease affecting ?10% of early-onset dementia patients. Using an enzyme-linked immunosorbent assay, we previously showed that GRN is detectable in human plasma and can be used to predict GRN mutation status. This study also showed a wide range in plasma GRN levels in non-GRN mutation carriers, including controls. We have now performed a genome-wide association study of 313,504 single-nucleotide polymorphisms (SNPs) in 533 control samples and identified on chromosome 1p13.3 two SNPs with genome-wide significant association with plasma GRN levels (top SNP rs646776; p = 1.7 × 10?³?). The association of rs646776 with plasma GRN levels was replicated in two independent series of 508 controls (p = 1.9 × 10?¹?) and 197 FTLD patients (p = 6.4 × 10?¹²). Overall, each copy of the minor C allele decreased GRN levels by ?15%. SNP rs646776 is located near sortilin (SORT1), and the minor C allele of rs646776 was previously associated with increased SORT1 mRNA levels. Supporting these findings, overexpression of SORT1 in cultured HeLa cells dramatically reduced GRN levels in the conditioned media, whereas knockdown of SORT1 increased extracellular GRN levels. In summary, we identified significant association of a locus on chromosome 1p13.3 with plasma GRN levels through an unbiased genome-wide screening approach and implicated SORT1 as an important regulator of GRN levels. This finding opens avenues for future research into GRN biology and the pathophysiology of neurodegenerative diseases.
| S-EPMC2997361 | biostudies-literature
Project description:BACKGROUND: Genetic as well as epigenetic alterations are a hallmark of both epithelial and haematological malignancies. High throughput screens are required to identify epigenetic markers that can be useful for diagnostic and prognostic purposes across malignancies. RESULTS: Here we report for the first time the use of the MIRA assay (methylated CpG island recovery assay) in combination with genome-wide CpG island arrays to identify epigenetic molecular markers in childhood acute lymphoblastic leukemia (ALL) on a genome-wide scale. We identified 30 genes demonstrating methylation frequencies of > or =25% in childhood ALL, nine genes showed significantly different methylation frequencies in B vs T-ALL. For majority of the genes expression could be restored in methylated leukemia lines after treatment with 5-azaDC. Forty-four percent of the genes represent targets of the polycomb complex. In chronic myeloid leukemia (CML) two of the genes, (TFAP2A and EBF2), demonstrated increased methylation in blast crisis compared to chronic phase (P < 0.05). Furthermore hypermethylation of an autophagy related gene ATG16L2 was associated with poorer prognosis in terms of molecular response to Imatinib treatment. Lastly we demonstrated that ten of these genes were also frequently methylated in common epithelial cancers. CONCLUSION: In summary we have identified a large number of genes showing frequent methylation in childhood ALL, methylation status of two of these genes is associated with advanced disease in CML and methylation status of another gene is associated with prognosis. In addition a subset of these genes may act as epigenetic markers across hematological malignancies as well as common epithelial cancers.
| S-EPMC2838813 | biostudies-other
Project description:To further understand the roles played by the essential phosphoinositide PI4,5P(2), we have used a synthetic lethal analysis, which systematically combined the mss4(ts) mutation, partially defective in PI4P 5-kinase activity, with each of approximately 4700 deletion mutations. This genomic screening technique uncovered numerous new candidate effectors and regulators of PI4,5P(2) in yeast. In particular, we identified Slm1 (Yil105c), a previously uncharacterized PI4,5P(2) binding protein. Like Mss4, Slm1 and its homolog Slm2 (Ynl047c) were required for actin cytoskeleton polarization and viability. Co-immunoprecipitation experiments revealed that Slm1 interacts with a component of TORC2, a Tor2 kinase-containing complex, which also regulates the actin cytoskeleton. Consistent with these findings, phosphorylation of Slm1 and Slm2 was dependent on TORC2 protein kinase activity, both in vivo and in vitro, and Slm1 localization required both PI4,5P(2) and functional TORC2. Together, these data suggest that Slm1 and Slm2 function downstream of PI4,5P(2) and the TORC2 kinase pathway to control actin cytoskeleton organization.
| S-EPMC522789 | biostudies-other
Project description:The mechanisms that coordinate the regulation of autophagy with developmental signaling during multicellular organism development remain largely unknown. Here, we show that impaired function of ribosomal protein RPL-43 causes an accumulation of SQST-1 aggregates in the larval intestine, which are removed upon autophagy induction. Using this model to screen for autophagy regulators, we identify 139 genes that promote autophagy activity upon inactivation. Various signaling pathways, including Sma/Mab TGF-? signaling, lin-35/Rb signaling, the XBP-1-mediated ER stress response, and the ATFS-1-mediated mitochondrial stress response, regulate the expression of autophagy genes independently of the TFEB homolog HLH-30. Our study thus provides a framework for understanding the role of signaling pathways in regulating autophagy under physiological conditions.
| S-EPMC4197881 | biostudies-other
Project description:A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting cellular host-factors that can modify prion propagation. We exposed prion-infected cells in high-density microplates to 35'364 ternary pools of 52'746 siRNAs targeting 17'582 genes representing the majority of the mouse protein-coding transcriptome. We identified 1191 modulators of prion propagation. While 1151 modified the expression of both the PrPSc pathological prion protein and its cellular counterpart, PrPC , 40 genes selectively affected PrPSc. Of the latter 40 genes, 20 augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC . Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Further, the unexpected identification of a prion-controlling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.
| S-SCDT-10_15252-EMBJ_2022112338 | biostudies-other
Project description:Regulation of cellular death is central to nearly all physiological routines and is dysregulated in virtually all diseases. Cell death occurs by two major processes, necrosis which culminates in a pervasive inflammatory response and apoptosis which is largely immunologically inert. As necrosis has long been considered an accidental, unregulated form of cellular death that occurred in response to a harsh environmental stimulus, it was largely ignored as a clinical target. However, recent elegant studies suggest that certain forms of necrosis can be reprogrammed. However, scant little is known about the molecules and pathways that orchestrate calcium-overload-induced necrosis, a main mediator of ischemia/reperfusion (IR)-induced cardiomyocyte cell death. To rectify this critical gap in our knowledge, we performed a novel genome-wide siRNA screen to identify modulators of calcium-induced necrosis in human muscle cells. Our screen identified multiple molecular circuitries that either enhance or inhibit this process, including lysosomal calcium channel TPCN1, mitophagy mediatorTOMM7, Ran-binding protein RanBP9, Histone deacetylase HDAC2, chemokine CCL11, and the Arp2/3 complex regulator glia maturation factor-? (GMFG). Notably, a number of druggable enzymes were identified, including the proteasome ?5 subunit (encoded by PSMB5 gene), which controls the proteasomal chymotrypsin-like peptidase activity. Such findings open up the possibility for the discovery of pharmacological interventions that could provide therapeutic benefits to patients affected by myriad disorders characterized by excessive (or too little) necrotic cell loss, including but not limited to IR injury in the heart and kidney, chronic neurodegenerative disorders, muscular dystrophies, sepsis, and cancers.
| S-EPMC7088983 | biostudies-literature
Project description:Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. Here, to identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide small interfering RNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to messenger RNA processing, interferon signalling, vesicle trafficking, cytoskeletal motor function and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, SMURF1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, SMURF1-deficient mice accumulate damaged mitochondria in the heart, brain and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines SMURF1 as a newly recognized mediator of both viral autophagy and mitophagy.
| S-EPMC3229641 | biostudies-literature